Spontaneous development of plasmacytomas in a selected subline of BALB/cJ mice.

Sixty per cent of BALB/cAnPt mice injected intraperitoneally (i.p.) with tetramethylpentadecane (pristane) develop plasmacytomas (PCs), whereas less than 10% of BALB/cJ develop such tumours. Most other mouse strains are completely resistant. Resistance is dominant over susceptibility in F1 hybrids between BALB/cAnPt and the resistant non-BALB/c strains, suggesting that susceptibility may be due to some genetic defect. (BALB/cAnPtxBALB/cJ)F1 hybrids have a PC incidence of 36-42%. Previously, BALB/cJ has been shown to harbour at least one resistance gene (Potter et al., Genomics 1988, Vol. 2, pp. 257-262). On the assumption that BALB/cJ may contain a segregating resistance gene, we cross BALB/cJ females with pristane-pretreated BALB/cJ males that were found to be carrying PC cells intraperitoneally 5-7 months after pristane treatment. After two selective crosses, 62% of the BALB/cJ subline BALB/cM2/22 developed PC after pristane and 52% after pristane followed by Abelson virus, while unselected controls had an incidence of 11% and 0%, respectively. Moreover, six spontaneous plasmacytomas developed in untreated females of the selected colony. Five of these carried T(12; 15) (F2; D2/3) translocations. The sixth had a T(1; 10) (G; C1) translocation and an interstitial duplication of segment (C1/E3) on one chromosome 5. It may be concluded that pristane treatment is not a prerequisite for the induction of the PC associated Ig/myc translocations.

Ig/myc translocations of the plasmacytoma-prone BALB/c strain occur independently of the genetic and parental origin of the affected chromosomes 6, 12, and 15.

Virtually all murine plasmacytomas (MPCs) carry chromosomal translocations that juxtapose myc on chromosome 15 (chr 15) to one of the three immunoglobulin loci carrying chr 6, 12, or 16. Only some mouse strains are susceptible to MPC induction, however, with BALB/c as the outstanding example. Most other strains are resistant. Our earlier studies with reciprocal BALB/c<-->DBA/2 chimeras suggested that part of this susceptibility is determined at the level of the target cell itself (DBA/2 is MPC resistant). The probability of the Ig/myc translocation is one of the possibly relevant variables. Because MPC resistance is dominant over susceptibility, it is conceivable that the translocations prevail due to some deficiency of the Ig rearrangement or Ig-associated repair mechanisms in BALB/c cells. This could be determined at the level of the chromosomes that participate in the translocation or by genes on other chromosomes. Here, we show that the substitution of the BALB/c-derived chr 12, 6, and 15, which carry IgH, kappa, and myc, respectively, with their homologs derived from MPC-resistant mice, did not affect MPC susceptibility. The use of Robertsonian 4.12 and 6.15 chromosomes in this study has also provided us with the opportunity to assess the parental derivation of the chromosomes participating in the translocation. In contrast to the human chronic myeloid leukemia (CML)-associated BCR/ABL fusion transcript, where a strong bias was claimed that was attributed to imprinting, we have found that the parental chromosomes were randomly involved in the translocation. We have also shown that the translocations could be of uniparental or biparental origin.

Hypersomy of chromosome 15 with retrovirally rearranged c-myc, loss of germline c-myc and IgK/c-myc juxtaposition in a macrophage-monocytic tumour line.

From a lymphoid tumour induced by 7,12-dimethylbenz-[a]-anthracene (DMBA) + methyl-N-nitrose-N-urea (MNU) in an [AKR Rb(6.15) x CBAT6T6]F1 mouse, a macrophage- monocyte line (KT-10) was isolated. Following ethyl methanesulfonate (EMS) treatment, a bromodeoxyuridine (BUdR) resistant subline was selected. Serial propagation of this line in vitro in the presence of BUdR (28 months) with periodic cytogenetic and molecular examinations, has led to the definition of four successive stages. During stage I, the cells were trisomic for chromosome 15. They contained Rb(6.15) and Rb(del6.15) of AKR and T(14;15) of CBA origin. Southern blotting showed the presence of both germline (G) and rearranged (R) c-myc. At stage II, Rb(del6.15) has duplicated. Both Rb(6.15) and T(14;15) persisted together with G-myc and R-myc. In stage III, the CBA-derived T(14;15) was lost, in parallel with G-myc. At this stage, a Dic.In(6.15) was detected. One of its arms was cytogenetically identical with the long arm of In(6.15) in the variant IgK/myc translocations. This chromosome carried R-myc and IgK in juxtaposition, as indicated by comigration on pulsed field electrophoresis (PFGE). At stage IV, the R-myc carrying AKR-derived chromosome 15s were present in six copies. Possible relationships between the increasing R/G myc ratio and changed growth characteristics in vivo and in vitro are discussed.

Correlation of myc expression with the growth-arrested and transformed phenotypes in hybrids between a T lymphoma and an antigen-responsive T-cell line.

Fusion of the YACUT lymphoma cell line with the Mls-1a-antigen-specific non-tumorigenic T-cell line G4 produced growth-arrested hybrids that could be induced to proliferate in the presence of Mls-1a antigen. Prolonged growth of such hybrids by repeated antigenic stimulation resulted in the appearance of autonomously growing hybrid lines. Of the 4 antigen-independent hybrid clones, I was weakly tumorigenic (25% incidence) while the other 3 were highly tumorigenic (100% incidence). In the growth-arrested hybrids the de-regulated c-myc expression characteristic of the YACUT cells was suppressed. In the autonomously growing clones, however, c-myc expression had reverted to the levels of the lymphoma parent and 1 to 2 extra copies of chromosome 15 were consistently present. These results indicate that repeated antigenic stimulation somehow abrogated the down-regulation of c-myc in the growth-arrested hybrid lines. The increase in the number of copies of chromosome 15, however, suggests that genes located on this chromosome may abolish the effect of the negative regulatory functions of the non-malignant parent in a gene-dosage-dependent manner.

Functional homology between N-myc and c-myc in murine plasmacytomagenesis: plasmacytoma development in N-myc transgenic mice.

Mouse plasmacytomas induced by pristane oil alone, or in combination with Abelson murine leukemia virus (A-MuLV), regularly carry one of three alternative chromosomal translocations that juxtapose c-myc to immunoglobulin heavy- or light-chain loci. E mu-c-myc transgenic mice develop translocation-free plasmacytomas after induction by pristane oil and/or A-MuLV [Sugiyama, H., Silva, S., Wang, Y., Weber, G., Babonits, M., Rosen, A., Wiener, F. & Klein, G. (1990). Int. J. Cancer, 46, 845-852]. In order to test whether another member of the myc family, N-myc, could play a similar role as c-myc, we treated E mu-N-myc transgenic mice with pristane and helper-free A-MuLV. Of 20 mice that received a single pristane injection followed by A-MuLV, 17 developed plasmacytomas with a mean latency period of 54 +/- 20 days. In a corresponding group that only received a single pristane injection, five out of six transgenic mice developed plasmacytomas with a mean latency period of 142 +/- 32 days. However, after three monthly injections of pristane, all 15 transgenic mice developed plasmacytomas with a mean latency period of 128 +/- 20 days. All plasmacytomas expressed the N-myc transgene, while none of them expressed either c-myc or endogenous N-myc. None of the tumors carried the usual plasmacytoma-associated translocations.

An exceptional mouse plasmacytoma with a new kappa/N-myc [T(6; 12) (C1; B)] translocation expresses N-myc but not c-myc.

Mouse plasmacytomas (MPC) carry one of three reciprocal translocations that juxtapose c-myc to one of the three immunoglobulin (Ig) loci. Here we describe an exceptional MPC, induced by pristane oil and Abelson (A-MuLV) virus. It does not carry any of the three c-myc/Ig translocations, but contains a previously unknown reciprocal T(6;12) translocation affecting the bands known to carry the IgK (6C/1) and N-myc (12B) loci, respectively. Northern blot analysis showed high N-myc but no c-myc expression. This is consistent with the constitutive activation of N-myc by a juxtaposition of the IgK and N-myc loci. Reciprocal translocation in B-cell derived tumors are believed to involve the Ig loci by the action of some enzyme that participates in the physiological rearrangement of the Ig loci. Only transcriptionally active chromatin regions are accessible to such recombinases (Alt et al. 1987). N-myc is not expressed in B-cells, but it is transcriptionally active during the early pro- and pre-B cell stage, whereafter it and the surrounding chromatin region becomes inactive (Smith et al. 1992). It is therefore most likely that the N-myc/Kappa translocation has arisen at an early stage of B-cell differentiation. This would imply that the myc/Ig translocations do not block B-cell differentiation. They also reaffirm the functional equivalence of N- and c-myc in relation to B-cell carcinogenesis, as shown by our previous work on tumor induction in N-myc transgenic mice (Wang et al. 1992).

Differential susceptibility of BALB/c and DBA/2 cells to plasmacytoma induction in reciprocal chimeras.

Reciprocal chimeras were generated between BALB/c and DBA/2 mice by inoculating newborn recipients of either strain with bone-marrow (BM) cells of the other through the periorbital vein. DBA/2 mice inoculated with the BALB/c with proven chimerism will be referred to as C----D, the reciprocal as D----C. The BALB/c cells carried a Robertsonian 6;15 (Rb6;15) chromosome marker to facilitate identification. The chimeric mice contained between 5% and 70% of donor cells when examined at 4 to 5 weeks of age. Six of 10 C----D developed plasmacytomas (MPC) after 3 x 0.5 ml monthly pristane treatment (incidence 60%) and 8 of 25 (incidence 32%) after 2 to 3 x 0.5 ml pristane followed by Abelson virus (A-MuLV) infection. Seven of 15 D----C developed MPC after pristane treatment (incidence 47%) and 4 of 17 after pristane + A-MuLV (incidence 24%). All tumors that have arisen in both reciprocal chimeras originated from BALB/c cells independently of the degree of chimerism. All tumors contained an Ig/myc translocation. Among the C----D chimeras, 5 carried t(12;15) and I t(6;15) in the pristane-treated group, while 4 carried t(12;15), I t(6;15) and 3 t(15;16) in the pristane + A-MuLV. Among the D----C chimeras 6 carried t(12;15) and I t(6;15) in the pristane-treated group, while 3 t(12;15) and I t(6;15) in the pristane + A-MuLV. No tumors developed in 18 pristane- and 22 pristane + A-MuLV-treated DBA/2 mice nor in 15 pristane- and 17 pristane + A-MuLV-treated (BALB/c x DBA/2)F1 mice. The data indicate that BALB/c and DBA/2 cells differ in their propensity to transform into plasmacytoma in identical host environments after both pristane and pristane + A-MuLV treatment. They also show that the oil granuloma can support MPC development in either type of chimeric host.

v-abl does not abolish IL-6 requirement by murine plasmacytoma cells.

Activation of c-myc by juxtaposition to an immunoglobulin locus and introduction of the v-abl oncogene act synergistically in generating a mouse plasmacytoma (PC). The question arose whether the effect of v-abl could be attributed to a deregulation of interleukin-6 (IL-6) production or responsiveness, in view of the fact that IL-6 exerts potent growth-stimulatory activity on PC cells. We studied the effect of IL-6 on the in vitro growth of primary PCs induced by pristane alone (TEPCs) or by pristane + A-MuLV (ABPCs). Five of 13 TEPCs and 3 of 7 ABPCs responded to IL-6. Macrophage supernatants prepared from both TEPCs and ABPCs had similar stimulatory effects on PC cells. From 30 primary PCs (including both TEPCs and ABPCs), we established 9 in vitro lines, 2 of which expressed v-abl. All were able to grow on macrophage feeder layers. Three types of behavior could be distinguished on the basis of growth in feeder-free cultures in the presence and absence of IL-6. Group I contained 4 IL-6-dependent lines. Group II contained 2 IL-6-independent lines (one v-abl expressor) that grew faster in the presence of IL-6. Group III consisted of 3 feeder-dependent lines (one v-abl expressor) that were not significantly stimulated by IL-6. These findings indicate that v-abl expression does not influence IL-6 dependence or responsiveness by itself. The supernatant of one line in group II was able to stimulate PC cells. All 6 lines of Groups I and II carried a typical (12;15) translocation, while all 3 lines in group III had a variant (6;15) or (15;16) translocation.

Abelson murine leukemia virus transforms preneoplastic Emu-myc transgene-carrying cells of the B-lymphocyte lineage into plasmablastic tumors.

E mu-myc transgenic mice were back-crossed to BALB/c mice up to back-cross generation 3. The offspring that included transgene-carrying and -negative mice in approximately equal proportions were randomly divided into 2 groups. Thirty-four mice (group I) were treated with pristane, followed by A-MuLV, and 40 (group II) were injected with A-MuLV alone. Altogether, 16 lymphoid tumors developed in group I and 17 in group II. Nine of the tumors in group I and 4 in group II appeared as ascitic tumors. The ascites contained lymphoblasts and 10 to 45% plasmacytoid cells. These tumors were designated as plasmablastic lymphomas (PLs). All tumors except one were transgene-positive and did not carry translocations. An exceptional tumor in group I carried a variant 6;15 translocation but not the transgene. It obviously corresponds to the regular Abelson + pristane-induced plasmacytoma. Among 11 tested PLs, 10 had a single retroviral insertion site, while one tumor showed 3. Among 18 untreated transgenic descendants (group III), chosen randomly during serial back-crosses, 15 (83%) developed lymphomas, with no sign of plasmacytoid differentiation. The incidence was comparable in all 3 groups, assuming 50% of the mice in groups I and II to be transgenic. The time distribution of tumor development was also similar. Spleen cells from transgene-carrying mice with no clinical sign of lymphoma were infected in vitro with A-MuLV and transplanted i.p. into BALB/c recipients. PLs developed in 26 of 31 pristane-treated recipients, but in only one of 18 untreated recipients. One of 6 PLs tested was monoclonal, whereas the remaining 5 were oligoclonal. They all expressed v-abl. These results show that some of the preneoplastic B-cells that expressed constitutively active myc transgene turned into plasmablasts after infection with A-MuLV. Full development of their neoplastic potential was facilitated by the presence of pristane-granuloma.

A murine plasmacytoma with a variant (15;16)(D2/3;B1) translocation that involves the c-myc and lambda light chain gene-carrying chromosomes.

A murine plasmacytoma (MPC) with a reciprocal translocation between chromosomes 15 and 16 with breakpoints in 15D2/3 and 16B1 is reported. The breakpoint on chromosome 15 is identical to the breakpoint in the MPC-associated typical (12;15) and kappa variant (6;15) translocation. Therefore it probably involves the c-myc gene as well. Unlike the Burkitt lymphoma (BL) system, a lambda/myc variant translocation has not been described in the MPC system. Chromosome 16 is known to carry the lambda gene. Therefore, the 15;16 translocation probably represents the "missing" lambda/myc variant in MPC, suggesting that the lambda gene is localized at 16B1.

The "missing" mouse plasmacytoma (MPC) associated translocation T(15;16) occurs repeatedly in new MPC induction systems.

A reciprocal translocation between chromosomes 15 and 16 has been detected in seven murine plasmacytomas induced by a combination of pristane and Abelson virus. Six of the tumors were induced in a new, unconventional experimental system based on transfer of uninfected or Abelson virus infected bone marrow and spleen cells, respectively, into pristane treated mice. All six tumors were of donor type. The seventh tumor appeared in a conventional pristane + Abelson virus treated mouse. This tumor was unusual in carrying both the 15;16 variant translocation and the typical 12;15 translocation, in the same tumor cells. In the new 15;16 variant, the breakpoint of chromosome 15 was at the interphase of the D2/3 sub-bands, as in mouse plasmacytomas with the previously well-known typical 12;15 and variant 6;15 translocations. The breakpoint on chromosome 16 was mapped to band 16B1, corresponding to the presumed cytogenetic site of the Ig-lambda gene. In three of the seven tumors with the 15;16 translocation, the derivative chromosome 15 had undergone a duplication, a feature that has not been previously encountered in the MPC-associated 12;15 and 6;15 translocation carriers. The reciprocal derivative chromosome 16 was lost from one of the seven tumors. We postulate that the 15;16 translocation results in juxtaposition of the myc gene to lambda sequences, probably in a similar orientation as previously described for the variant 6;15 translocation.

The accelerating role of Abelson murine leukemia virus in murine plasmacytoma development: in vitro infection of spleen cells generates donor-type tumors after transfer to pristane-treated BALB/c mice.

The role of Abelson murine leukemia virus (A-MuLV) in the accelerated development of murine plasmacytomas (PCs) (Potter et al., 1973: Science, 132, 592-594) was studied in a new experimental system. Spleen cells from pristane-treated or untreated BALB/c mice carrying Robertsonian 6;15 fusion chromosomes were infected in vitro with helper-free A-MuLV overnight and subsequently transplanted into the peritoneal cavity of pristane-treated or untreated BALB/c mice. Donor-derived PCs developed in 4 out of 76 pristane-treated recipients [latent periods: 38-82 (mean 51) days] that had received spleen cells from pristane-treated donors, and also in 2 out of 41 pristane-treated recipients that had received untreated donor-derived spleen cells (latent periods: 65 and 120 days). Three of the PCs in the former and both PCs in the latter group were tested for integration and expression of the v-abl gene, with positive results. This indicates that the spleen contains PC-precursor cells that can be activated by A-MuLV even before the impact of pristane. All 6 donor-origin PCs carried a translocation involving chromosome 15, band D2/3. Four of these corresponded to a typical 12;15 translocation, one was a variant 6;15 translocation and the 6th may represent a previously unidentified translocation between chromosome 15 and the lambda gene-carrying chromosome 16. No PCs developed among 29 pristane-untreated recipients that had received pristane-treated donor-derived spleen cells. In addition to PCs, monocytic tumors developed in 37 (26%) of all recipients. Their development was independent of pristane treatment of recipients but was particularly frequent in those who had received spleen cells from pristane-treated donors.

Plasmacytoma induction in radiochimeras.

We have induced plasmacytomas (MPC) in BALB/c radiochimeras (RCh) repopulated with syngeneic hemopoietic cells carrying distinctive chromosomal markers. A group of 38 RCh that received 0.5 ml pristane, followed by Abelson virus infection 2-3 weeks later, developed 7 tumors (18.5%) of donor origin after a relatively short latency period (X = 83 +/- 8.3 days). In contrast, only 3 (2%) MPCs were observed in 149 RChs that received 0.5 ml pristane 3 times at monthly intervals. Two of them originated from host cells. Pristane-treated RChs developed a less extensive oil granuloma (OG), compared with pristane-treated intact mice. This may explain the low incidence of MPC in the former. Our findings also suggest that Abelson virus can overcome the postulated deficiency of OG. MPC induction in the pristane + Abelson-virus-treated RCh system will facilitate the further characterization of the MPC precursor cell and the localization of genetic resistance vs. susceptibility factors at the donor vs. host level.

Further studies on chromosome 15 trisomy in murine T-cell lymphomas: mapping of the relevant chromosome segment.

Trisomy 15 is the most common chromosomal aberration in murine T-cell lymphomas. The relevant chromosomal region responsible for the growth advantage of the 15-trisomic cell has not been defined. In order to map this region, we have induced thymic lymphomas by chemical carcinogens (DMBA or MNU) in mice with 2 different constitutional translocations, T(7;15)9H homozygotes and [T(7;15)9H X T(5;15)4Ad] FI hybrids. Twenty-two tumors developed in 90 carcinogen-treated mice. Among the 14 cytogenetically analyzed thymic lymphomas, 4 were diploid and 5 were aneuploid, with no chromosome-15-associated changes. Five lymphomas showed partial duplication of chromosome 15. Four of them have duplicated the segment distal to the C/DI breakpoint of T9H, while the 5th carried an interstitial duplication of the D2 sub-band of the T(7;15) translocation chromosome. These findings suggest that the duplication of the D 2/3 region, known to contain the c-myc and the pvt-I genes (Banerjee et al., 1985), rather than other regions of chromosome 15, contributes to the development and/or progression of murine T-cell leukemias.

The role of chromosome 15 in murine leukemogenesis. II. Relationship between tumorigenicity and the dosage of lymphoma vs. normal-parent-derived chromosomes 15 in somatic cell hybrids.

Somatic cell hybrids were generated between an MCF-virus-induced 15-trisomic T-cell lymphoma of AKR origin with a proviral insertion near the c-myc locus, and normal diploid fibroblasts or lymphocytes of CBAT6T6 origin. Three lymphoma/fibroblast fusions were performed. Six independently-derived clones from 2 fusions were tested for tumorigenicity. Three of the 6 clones were weakly malignant (take incidence 20% below), and 3 were strongly malignant (take incidence over 80%). All 3 lymphoma/lymphocyte hybrids and 6 derived clones were strongly malignant. All hybrids contained a nearly complete chromosomal complement of both parental cells. This was confirmed at the molecular level by determining the ratio of germ-line (G) vs. rearranged (R) myc-carrying Eco RI fragments that showed the expected 1.9-2.7:1 proportion. Malignant segregants selected from the weakly malignant lymphoma/fibroblast hybrids by in vivo inoculation showed changed 15-chromosome ratios. Four out of the 6 clones showed amplification of the lymphoma-derived 15-chromosome that carries the R-myc fragment and a concomitant decrease in the average number of the G-myc-carrying chromosomes. This was deduced from the fact that the G:R ratio was between 2 and 3:1 in the in vitro hybrids but became inverted (1:2-3) in the tumors. Two tumors showed no amplification of R-myc. G-myc was decreased. One of these tumors showed a change in the G:R ratio from 2.5:1.0 to 1.2:1.0, while the other was essentially unchanged (1.9:1.0 in the in vitro clone and 2.2:1.0 in the derived tumor). These findings support the notion that both the amplification of the lymphoma-derived 15-chromosome with the retrovirally rearranged c-myc carrying fragment and/or the loss of the G-myc-carrying 15-chr can contribute to the tumorigenic potential of the hybrids.

Avian v-myc replaces chromosomal translocation in murine plasmacytomagenesis.

Deregulation of c-myc expression in association with chromosomal translocations occurs in over 95% of murine plasmacytomas, rat immunocytomas, and human Burkitt lymphomas. Infection with a murine retrovirus (J-3) containing an avian v-myc rapidly induced plasmacytomas in pristane-primed BALB/cAn mice. Only 17% of the induced plasmacytomas that were karyotyped showed the characteristic chromosomal translocations involving the c-myc locus. Instead, all of the translocation-negative tumors demonstrated characteristic J-3 virus integration sites that were actively transcribed. Thus, the high levels of v-myc expression have replaced the requirement for chromosomal translocation in plasmacytomagenesis and accelerated the process of transformation.

Mapping of the c-myc, pvt-1 and immunoglobulin kappa genes in relation to the mouse plasmacytoma-associated variant (6;15) translocation breakpoint.

A variant mouse plasmacytoma (MPC)-associated translocation chromosome has arisen by pericentric inversion and exchange of the distal segments of a Robertsonian 6;15 fusion chromosome in the CAK TEPC 1198 mouse plasmacytoma, as described earlier. In situ hybridization was performed on the normal and the inverted Rb chromosomes, using myc and kappa probes. On the normal Rb chromosome, myc was in the 15 D2/3 region, whereas kappa hybridized in the 6 C2 area, as expected. On the inverted Rb chromosome, myc remains on the centrometric side of the translocation breakpoint on the chromosome 15-derived portion, whereas kappa has moved to the chromosome 6-derived segment that joined the same breakpoint on the telomeric side. Taken together with our recent demonstration that the murine c-myc locus is oriented 'head up' on chromosome 15, and with the results of Cory and co-workers concerning the relationship between the kappa gene and the associated pvt-1 region in the CAK TEPC 1198 tumor, the following conclusions can be drawn: (i) in the variant translocation of the CAK TEPC 1198 MPC, the breakage occurs 3' of the c-myc gene, as in the human Burkitt lymphoma-associated variant translocations; (ii) the pvt-1 gene on chromosome 15 is distal to the myc gene; (iii) the kappa light chain locus is oriented 'head up' on mouse chromosome 6 and faces pvt-1 and, beyond it, c-myc, in a head-to-tail configuration.